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Journal of Fish Biology (2009) 75, 2326–2343
doi:10.1111/j.1095-8649.2009.02489.x, available online at www.interscience.wiley.com




       Molecular and karyotypic phylogeography in the
      Neotropical Hoplias malabaricus (Erythrinidae) fish
                       in eastern Brazil

                              ¨
           U. Santos*, C. M. Volcker†, F. A. Belei*, M. B. Cioffi‡,
            L. A. C. Bertollo‡, S. R. Paiva§ and J. A. Dergam*
      *Laborat´ rio de Sistem´ tica Molecular Beagle, Departamento de Biologia Animal,
              o              a
 Universidade Federal de Vi¸ osa, 36570-000 Vi¸ osa, MG, Brazil, †Rua Edgard Coutens de
                             c                  c
 Menezes, 47. 28970-000 Pontinha, Araruama, RJ, Brazil, ‡Centro de Ciˆ ncias Biol´ gicas
                                                                         e         o
 e da Sa´ de, Departamento de Gen´ tica e Evolu¸ ao, Universidade Federal de S˜ o Carlos,
          u                          e            c˜                            a
  S˜ o Carlos, SP, Brazil and §Embrapa Recursos Gen´ ticos e Biotecnologia, Laborat´ rio
   a                                                   e                             o
Gen´ tica Animal, PCG, Parque Esta¸ ao Biol´ gica, Final Av. W/5 Norte, Bras´lia, DF, Brazil
   e                                 c˜      o                               ı

                          (Received 9 June 2009, Accepted 13 October 2009)

     The sedentary, predatory characin Hoplias malabaricus has one of the widest distributions of fresh-
     water fishes in South America and is characterized by seven karyomorphs (A–G) that occur in
     sympatric and allopatric populations. Karyotypical patterns of variation in wild populations have
     been interpreted as evidence of multiple lineages within this nominal species, a possibility that may
     limit the validity of experimental data for particular karyomorphs. This study used the phylogeo-
     graphic and genealogical concordance between cytogenetic (N = 49) and molecular (mitochondrial
     DNA) (N = 73) data on 17 samples, collected in 12 basins from south-eastern and north-eastern
     Brazil, to assess the systematic value of cytogenetic data. Cytogenetic patterns show a sex chro-
     mosome system in the 2n = 40F karyomorph. Molecular and cytogenetic data indicate a long,
     independent evolutionary history of karyomorphs and a coastal origin of continental populations
     in south-eastern Brazil. The lack of fit with molecular clock expectations of divergence between
     groups is likely to be due to strong demographic fluctuations during the evolution of this species
     complex. The results indicate that karyotypical identification provides a reliable baseline for placing
     experimental studies on Hoplias spp. in a phylogenetic context.                      © 2009 The Authors
                                                     Journal compilation © 2009 The Fisheries Society of the British Isles

     Key words: freshwater fish; in situ hybridization; molecular clock; mtDNA; repetitive DNAs; South
                America.




                                           INTRODUCTION
The Neotropical region harbours at least 6025 species of freshwater fishes (Reis
et al., 2003), but perhaps as many as 8000 (Schaefer, 1998), the result of a long evo-
lutionary history of isolation and specialization, involving mainly otophysan fishes.
Freshwater fishes are highly suitable for the recovery of past biogeographic processes,
due to their obligatory relationship with water (Myers, 1938). The biogeographical

    Author to whom correspondence should be addressed. Tel.: +55 31 3899 2555; fax: +55 31 3899 2578;
email: dergam@ufv.br

                                                     2326
                                                                                                 © 2009 The Authors
                                                  Journal compilation © 2009 The Fisheries Society of the British Isles
PHYLOGEOGRAPHY OF HOPLIAS MALABARICUS                                                         2327

information relevant for fish diversification has been reviewed by Lundberg et al.
(1998) and Ribeiro (2006). The former revision focused on the evolution of Andean
geomorphology and the effects of this process on the South American continent,
whereas the latter work dealt with the evolution of particular coastal drainages,
their geomorphological interactions with continental basins and the expected lev-
els of phylogenetic divergence among fishes derived during these complex temporal
events.
   Among Neotropical freshwater fishes, the trahira, Hoplias malabaricus (Bloch),
has one of the largest distributional ranges, occurring from Panam´ to the Buenos
                                                                       a
Aires Province in Argentina (Berra, 2007). H. malabaricus is well adapted to life
in small, isolated populations, in conditions that may facilitate the stochastic fixa-
tion of chromosomal rearrangements (Sites & Moritz, 1987). This species is one of
the cytogenetically most studied taxa and shows a conspicuous karyotypic diversi-
fication, with up to seven karyomorphs with diploid numbers ranging from 39 to
42. Three groups of karyomorphs are readily evident, based on distribution ranges.
Two karyomorphs, 2n = 42A and 2n = 40C, are the most widespread and occur in
sympatry in the lower Paran´ –Paraguay and Amazon River basins. A second, less
                              a
widely spread group is represented by three karyomorphs: (1) 2n = 40F occurs in
the S˜ o Francisco River basin, in the lower portions of the Tocantins River, in coastal
      a
drainages in Surinam and in minor basins in north-eastern Brazil; (2) 2n = 39/40D
occurs in the upper Paran´ River (in sympatry with 2n = 42A) and (3) 2n = 40/41G
                          a
occurs exclusively in the Amazonian Basin in the Aripuan˜ , Madeira and Trombetas
                                                             a
Rivers.
   The third group consists of karyomorphs with much restricted distributions. For
example, 2n = 42B occurs in the Doce River basin (also in sympatry with
2n = 42A), and 2n = 42E occurs only in the Trombetas River (Bertollo et al., 2000).
   The lack of hybrids between sympatric karyomorphs has been interpreted as evi-
dence for the existence of several distinct species within this nominal taxon (Bertollo
et al., 1986, 2000). Therefore, uniparental molecular markers can be a particularly
useful complement to assess gene flow in wild populations. To date, molecular stud-
ies on H. malabaricus have been restricted to populations in the Doce River basin
(Dergam et al., 2002) and the Iguacu River basin (Dergam et al., 1998). Both studies
                                    ¸
indicated phylogenetically related populations in the coastal and continental basins,
but failed to reveal the direction of dispersal events.
   Karyotypic and molecular patterns of variation may reveal vicariances and dis-
persals that provide insights into evolutionary processes that may involve other
components of the aquatic fauna. The congruence of independent molecular and
cytogenetic characters may give support to the hypothesis that at least some kary-
omorphs behave as valid species within H. malabaricus. A better understanding of
H. malabaricus species-level systematics is needed to interpret correctly the compar-
ative physiological, behavioural and anatomical studies in an explicit phylogenetic
framework (Harvey & Pagel, 1991). Biodiversity patterns in this species complex
also bear potential information to understand the palaeohydrology of the continent. In
the present study, patterns of cytogenetic and molecular variation of H. malabaricus
populations in the S˜ o Francisco, Grande and in several coastal basins along eastern
                      a
Brazil were analysed using molecular and cytogenetic techniques.

© 2009 The Authors
Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
2328                                             U. SANTOS ET AL.


                                   MATERIALS AND METHODS

S P E C I M E N S A N D C H R O M O S O M E P R E PA R AT I O N
   Cytogenetic analyses were carried out on 49 specimens collected in the Pandeiros River,
Par´ River and its headwaters in the Tombadouro Creek and in the Jacar´ River (Table I
    a                                                                       e
and Fig. 1). Cell division was stimulated in vivo with two daily applications of Munolan, a
commercially available antigen lysate, following Molina (2001). Mitotic chromosomes were
obtained from cell suspensions of the anterior kidney, using the conventional air-drying
method (Bertollo et al., 1978). Fish were previously anaesthetized with clove oil (Henyey
et al., 2002). Secondary data from 10 populations with known chromosome numbers were
also included in the analysis.

C H R O M O S O M E S TA I N I N G
   In addition to the standard Giemsa method, chromosomes were analysed using silver nitrate
staining (Howell & Black, 1980) to visualize the nucleolar organizing regions (Ag-NOR).
C-banding was also employed to detect C-positive heterochromatin (Sumner, 1972). (4 ,6
diamidino-2-phenylindole dihydrochloride (DAPI) and chromomycin A3 (CMA3 ) fluores-
cence staining were used to identify the chromosome adenine–thymine (AT) and guanine-
cytosine (GC) rich regions, respectively (Sola et al., 1992). Some populations with unknown
karyotypes (or whose karyotypes are being studied by J.A.D.) were also included in this study
(see below).

C H R O M O S O M E H Y B R I D I Z AT I O N A N D K A RY O T Y P I C
A N A LY S I S
   Fluorescent in situ hybridization (FISH) was performed according to Pinkel et al. (1986)
using three repetitive DNA sequences probes isolated from the genome of H. malabaricus and
an 18S probe. The first probe contained a 5S rDNA repeat copy and included 120 base pairs


Table I. Hoplias malabaricus collecting localities, sample sizes, geographic coordinates and
                               karyomorph nomenclature

                             Cytogenetic Molecular
Locality                      samples     samples                             G.P.S.                    Karyomorph
Par´ River
    a                           8♀ –7♂              14        20◦   08   21   S–44◦     53   17    W        40F
Pandeiros River                 2♀ –3♂               9        15◦   40   18   S–44◦     37   43    W        40F
Tombadouro Creek                4♀ –3♂               5        20◦   41   56   S–44◦     34   46    W        42A
Ibimirim                          —                  6         8◦   30   31   S–37◦     42   21    W      Unknown
Curvelo                           12                 8        18◦   42   57   S–44◦     25   56    W        40F
Itapicuru River                     4                1        13◦   14   40   S–41◦     23   34    W        40F
Dom Helv´ cio Lake
              e                   10                 3        19◦   46   34   S–42◦     35   19    W        42B
S˜ o Mateus River
  a                                 2                6        18◦   44   09   S–48◦     29   47    W        42A
Para´ba do Sul River
      ı                           15                 2        21◦   46   26   S–41◦     30   01    W        42A
Itabapoana River                    8                2        21◦   08   20   S–41◦     39   35    W        42A
Maca´ River
        e                           6                1        22◦   17   43   S–41◦     52   48    W        42A
S˜ o Jo˜ o River
  a       a                         8                2        22◦   33   29   S–42◦     07   21    W        42A
Ribeira River                       2                1        24◦   29   23   S–47◦     50   10    W        42A
Carioca Lake                      15                 1        19◦   45   32   S–42◦     37   15    W        42B
Paranagu´ Bay
            a                     —                  1        25◦   32   39   S–48◦     29   47    W      Unknown
Macacu Waterfalls                 —                  1        22◦   27   37   S–42◦     39   18    W      Unknown
Jacar´ River
       e                       15♀ –7♂              10        20◦   48   29   S–44◦     33   58    W        42A


                                                                                                       © 2009 The Authors
       Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
PHYLOGEOGRAPHY OF HOPLIAS MALABARICUS                                                          2329

 Equator



                                                                                          35° 44′ 58′′ W
                                                                                                  8° 08′ 01′′ S
                                                                                             1
                                      500 km                                                       1 - Ibimirim
                                                                                      2            2 - Itapicuru River
                                                                                                   3 - Pandeiros River
                                                                   3
                                                                                                   4 - Buranhém River
                                                                                                   5 - Curvelo
                                                                                                   6 - Três Marias
                                                                                                   7 - São Mateus River
                                                                                      4            8 - Dom Helvécio and
                                                                                                       Carioca lakes
                                                             5                                     9 - Pará River
                                                         6                        7               10 - Tombadouro Creek
                                                                       8
                                                         9                                        11 - Itabapoana
                                                              10
                                                             12              11                   12 - Jacaré River
                                                                            13                    13 - Paraíba do Sul River
                                                                       14                         14 - Macacu
                                                             16 15                                15 - Macaé
                                                    17
                                               18                                                 16 - São João River
      26° 19′ 17′′ S                                                                              17 - Ribeira River
               54° 35′ 13′′ W                                                                     18 - Paranaguá

Fig. 1. Hoplias malabaricus collection localities. The dotted line indicates the upper Grande crustal disconti-
      nuity. The locality of Trˆ s Marias is also depicted.
                               e


(bp) of the 5S rRNA encoding gene and 200 bp of the non-transcribed spacer (NTS) (Martins
et al., 2006). The second probe is specific to H. malabaricus (Ferreira et al., 2007) and
contained a copy of the repetitive satellite 5SHind III-DNA sequence with 360 bp composed
of a 95 bp segment similar to the 5S rRNA gene of the first probe and a 265 bp segment similar
to the NTS of the first probe (Martins et al., 2006). The third probe corresponded to a 1400 bp
segment of the 18S rRNA gene obtained by the polymerase chain reaction (PCR) from
nuclear DNA (Cioffi et al., 2009). The probes were labelled by nick translation with biotin-14-
dATP (Bionick Labeling System, Invitrogen; www.invitrogen.com). Signal detection and its
amplification were performed using conjugated avidin–fluorescein isothiocyanate (FITC) and
anti-avidin–biotin (Sigma; www.sigmaaldrich.com). The chromosomes were counterstained
with propidium iodide (50 μg ml−1 ) and analysed with an Olympus BX50 epifluorescence
microscope. The chromosomal images were captured using CoolSNAP-Pro software (Media
Cybernetics; www.MediaCy.com). About 30 metaphase spreads were analysed per specimen
to determine the diploid chromosome number and karyotypic structure. Chromosomes were
classified as metacentrics (m) or submetacentrics (sm), according to centromeric index values
proposed by Levan et al. (1964).


D N A E X T R A C T I O N A N D M O L E C U L A R A N A LY S I S
   DNA extraction followed Boyce et al. (1989). Fragments of ATPase 6 were amplified
using primers L8524 (5 -AAY CCT GAR ACT GAC CAT G-3 ) and H9236 (5 -GTT AGT
GGT CAK GGG CTT GGR TC-3 ) (Quenouille et al., 2004). Double-stranded DNA was syn-
thesized in 50 μl reactions containing 10 μl dNTPs (1 mM), 5 μl reaction buffer (200 mM
Tris–HCl pH 8·4, 500 mM KCl), 2 μl MgCl2 (50 mM), 2 μl of each primer (10 mM), 0·5 μl
(2·5 U) Taq DNA polymerase (Phoneutria), 2 μl template DNA (100 ng/μl) and 26·5 μl H2 O.
PCR conditions were as follows: 94◦ C (2 min), five cycles of 94◦ C (45 s), 54◦ C (45 s) and
72◦ C (1·5 min) and 29 cycles of 94◦ C (45 s), 58◦ C (45 s) and 72◦ C (1·5 min). PCR
products were purified using Qiaquick (Qiagen; www.quiagen.com): 5 μl of the purified
PCR product were used in a 10 μl cycle sequencing reaction using a drhodamine terminator

© 2009 The Authors
Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
2330                                             U. SANTOS ET AL.

cycle sequencing kit (PE Applied Biosystems; www.appliedbiosystems.com). Sequences were
aligned using CLUSTAL X 1.83 in MEGA 4.0 (Tamura et al., 2007). Phylogenetic trees were
constructed with neighbour joining (NJ) (Saitou & Nei, 1987), maximum parsimony (MP),
maximum likelihood (ML) (Felsenstein, 1981) and Bayesian inference (MB) (Huelsenbeck &
Ronquist, 2001). The model of molecular evolution that best fitted the data was chosen using
Modeltest 3.7 (Posada & Crandall, 1998), and haplotype divergence was estimated within
and between haplogroups using this selected model. Phylogenetic signal in NJ, MP and ML
trees was assessed using bootstraps with 1000 repetitions. Bayesian inference was performed
with five million Markov-Chain Monte-Carlo (MCMC) steps to produce posterior probabil-
ities of nodes in the tree with MrModeltest 2 (Nylander, 2004). Hoplias lacerdae Miranda
Ribeiro was used as an outgroup. Sequences were deposited in GenBank (accession numbers
GQ848606–GQ848642).


                                                    RESULTS

K A RY O T Y P I C D ATA
Karyomorph 2n = 40F
   All specimens from the Pandeiros and Par´ rivers (except those from Par´ ’s
                                                  a                                a
headwaters, in Tombadouro Creek) had 2n = 40 chromosomes for both sexes. The
karyotypes were composed of 10 m + 10 sm pairs, without morphologically differ-
entiated sex chromosomes using Giemsa conventional staining [Fig. 2(a)], and were
typical 2n = 40F karyomorphs. C-banding showed heterochromatic blocks in cen-
tromeric regions, except for chromosome pairs 9 and 10, which had less conspicuous
blocks, in addition to faint telomeric marks in some chromosome pairs. Males always
showed an interstitial heterochromatic block in the short arms of one homologue of
the first chromosome pair [Fig. 2(c)], whereas females lacked this block [Fig. 2(e)].
This heterochromatic region was negative for DAPI staining but failed to show
fluorescence with CMA3 . A proximal heterochromatic block located on the short
arms of a small submetacentric pair was the only observed GC-rich segment in both
populations [Fig. 3(f), (i)]. Ag-NORs were telomeric and their numbers were either
fixed (four in the Par´ River) or varied from 4 to 5 (Pandeiros River) [Fig. 4(a), (b)].
                      a
FISH analyses with the repetitive sequences showed no differences between these two
populations. The 18S rDNA probe hybridized on the pericentromeric region of one
metacentric pair and on the telomeric regions of two submetacentric pairs [Fig. 3(a)].
The 5S rDNA probe hybridized on the pericentromeric region of a medium-sized
metacentric pair [Fig. 3(c)]. The repetitive 5SHind III-DNA was mapped in the cen-
tromeric region of 10 chromosome pairs [Fig. 5(a)]. Although Ibimirim samples were
not karyotyped, they were collected in a region with populations characterized by
2n = 40F karyomorph and were assumed to share this karyomorph.

Karyomorph 2n = 42A
   In the headwaters of Tombadouro Creek, all specimens had 2n = 42 chromosomes,
with a karyotype consisting of 11 m + 10 sm pairs and without morphologically dif-
ferentiated sex chromosomes [Fig. 2(b)], and were considered to be typical 2n = 42A
karyomorphs. Besides some telomeric marks, conspicuous heterochromatic bands
were found in the centromeric region of all chromosomes, except for chromosome
pairs 6, 19 and 20, which showed faint blocks [Fig. 2(d)]. Ag-NORs varied among
and within individuals (one to two pairs), and bitelomeric NORs were evident in
either one or two chromosomes [Fig. 4(c)]. In this population, the 18S rDNA probe

                                                                                                       © 2009 The Authors
       Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
PHYLOGEOGRAPHY OF HOPLIAS MALABARICUS                                                       2331

(a)                                                           (b)

                                                                m
  m
        1     2     3     4     5    6     7     8    9               1     2      3     4    5     6     7     8     9

       10                                                             10    11

 sm                                                            sm
       11    12    13    14    15    16   17    18   19               12    13    14    15    16    17    18    19    20

       20                                                             21
(c)                                                           (d)


  m     1      2     3    4     5    6     7    8    9          m
                                                                      1      2    3     4     5     6     7     8     9

        10                                                            10    11

 sm
                                                               sm
        11    12    13    14   15   16    17   18    19               12    13    14    15    16    17   18     19    20

        20                                                            21
(e)                                                           (f)


                                                                m
  m     1     2      3     4    5     6    7    8    9                1     2     3     4      5    6     7     8     9

        10                                                            10    11


 sm
                                                               sm
        11    12    13    14   15    16   17    18   19
                                                                      12    13   14    15     16    17   18     19    20
        20
                                                                      21                                       5 μm

Fig. 2. Giemsa-stained and C-banded karyotypes of Hoplias malabaricus. (a) conventional Giemsa-stained
      karyotype of populations from Pandeiros and Par´ rivers, (b) samples from Jacar´ River and Tombadouro
                                                      a                              e
      Creek, (c) C-banded karyotypes of males from Par´ and Pandeiros Rivers, (e) C-banded karyotypes of
                                                         a
      females from Pandeiros and Par´ Rivers, (d) C-banded karyotype of specimens from Tombadouro Creek
                                     a
      and (f) C-banded karyotype from Grande River. Arrows indicate an interstitial heterochromatic block in
      the short arm of a homologue of the first chromosome pair. This block appeared in males of Pandeiros
      and Par´ River populations, but not in females.
              a



hybridized on three chromosome pairs, with interstitial, telomeric and bitelomeric
sites [Fig. 3(b)]. The 5S rDNA probe hybridized on the interstitial region of a large
submetacentric pair [Fig. 3(d)]. The repetitive 5SHind III-DNA probe bound to the
centromeric region of nine chromosome pairs [Fig. 5(b)].
   Specimens from the Jacar´ River in the Grande drainage had 2n = 42 kary-
                              e
omorphs composed of 11 m + 10 sm pairs, without morphologically differentiated
sex chromosomes [Fig. 2(b)], and represented a typical 2n = 42A karyomorph. Het-
erochromatic blocks were centromeric in most chromosomes, except for pairs 6,
8, 11, 19 and 20, which showed faint bands, in addition to telomeric marks in
some chromosome pairs [Fig. 2(f)]. Ag-NORs were restricted to telomeric regions
of two chromosomes [Fig. 4(d)]. Fluorescent regions hybridized with 18S rDNA
and 5SHind III-DNA and showed a pattern similar to the Tombadouro Creek sample.

© 2009 The Authors
Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
2332                                              U. SANTOS ET AL.


          (a)                               (b)                               (c)




          (d)                               (e)                               (f)




          (g)                               (h)                               (i)




Fig. 3. Metaphase chromosome spreads of Hoplias malabaricus after FISH with 18S and 5S rDNA probes and
      DAPI–Chromomycin A3 staining. Numerical and positional variation of 18S rDNA sites in populations
      from (a) Pandeiros and Par´ Rivers, (b) Tombadouro Creek and Jacar´ River. Mapping of 5S rDNA sites
                                  a                                        e
      for populations of (c) Pandeiros and Par´ Rivers, (d) Tombadouro Creek and (e) Jacar´ River. DAPI
                                               a                                              e
      staining demonstrating absence of signals in males (f) and females (h) from Par´ River. Arrow indicates
                                                                                     a
      the interstitial heterochromatic block negative for DAPI in one homologue of the first chromosome
      pair in males. Sequential CMA3 staining showing GC-rich DNA segments located on a submetacentric
      chromosome pair on (g) males and (i) females. Bar = 5 μm.



Additionally, the 5S rDNA probe hybridized on the interstitial region of a small
metacentric pair [Fig. 3(e)]. As was the case with the Ibimirim samples, Paranagu´
                                                                                 a
and Macacu samples were collected within the range of 2n = 42A populations
(J. A. Dergam, unpubl. data) and were therefore assumed to be derived from these
populations.

M O L E C U L A R D ATA
   Multiple sequences 511 bp in length yielded 86 phylogenetically informative sites.
Translation of these sequences into the corresponding amino acid sequences resulted
in eight phylogenetically informative sites. The transitions:transversions ratio was
6·4, indicating that substitution rates were not saturated. The best model fit esti-
mated with Modeltest and MrModeltest was HKY+G, which was used for NJ.
In the tree, haplotypes were separated into two large clades with variable boot-
strap support (Fig. 6). The largest bootstrap values were obtained for haplogroup I,
which was composed of the 2n = 42A karyomorph from Tombadouro Creek, Ribeira,
Paranagu´ and Macacu rivers (Fig. 1). Haplogroup II was less supported and included
         a
all remaining H. malabaricus (N = 67), regardless of diploid number. Within this

                                                                                                       © 2009 The Authors
       Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
PHYLOGEOGRAPHY OF HOPLIAS MALABARICUS                                                         2333

           (a)                                               (b)




                                              5 μm                                                  5 μm


           (c)                                               (d)




                                                   5 μm                                              5 μm


Fig. 4. Metaphase chromosome spreads of Hoplias malabaricus after silver staining, showing Ag-NORs
      in populations from (a) Par´ River, (b) Pandeiros River, (c) Tombadouro Creek and (d) Jacar´ River.
                                 a                                                               e
      Arrows and arrowhead indicate telomeric and bitelomeric Ag-NORs, respectively.


  Table II. Within- and between-haplogroup molecular distances in Hoplias malabaricus

                                           Haplogroup I,              Haplogroup IIA,               Haplogroup IIB,
                                             2n = 42                     2n = 40                       2n = 42
Haplogroup I, 2n = 42                           0·032
Haplogroup II A, 2n = 40                        6·82                        0·0053
Haplogroup II B, 2n = 42                        9·20                        9·50                            2·65



haplogroup, two subclades were highly supported: haplotypes derived from popula-
tions with the 2n = 40F karyomorph (haplogroup IIA) and from populations with
2n = 42A and 2n = 42B karyomorphs (haplogroup IIB). Haplogroup IIA included
all 2n = 40 S˜ o Francisco River H. malabaricus, plus the Itapicuru River specimen.
               a
Haplogroup IIB included two well-defined lineages: one included haplotypes from
the Doce and S˜ o Mateus Rivers with no representatives in the Grande River, and
                  a
a second lineage indicated a close phylogenetic relatedness between H. malabaricus
from the Jacar´ (Grande River), the Tombadouro (S˜ o Francisco River) and the
                 e                                      a
Maca´ , S˜ o Jo˜ o, Itabapoana and Para´ba do Sul coastal basins. Within-haplogroup
      e a       a                       ı
nucleotide molecular distances showed high levels of variation, with the smallest val-
ues in the S˜ o Francisco and Itapicuru samples and the largest in the IIB haplogroup.
            a
The range of between-haplogroup variation was much smaller (Table II).

© 2009 The Authors
Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
2334                                             U. SANTOS ET AL.


                   (a)



                           m
                                   1       2      3      4      5      6       7     8        9

                                  10


                          sm      11      12     13     14     15      16      17        18   19


                                  20

                  (b)


                           m
                                  1       2      3      4       5      6      7      8        9

                                 10      11


                          sm
                                 12      13      14     15     16     17     18       19      20


                                  21



Fig. 5. Karyotypes of Hoplias malabaricus samples from (a) Par´ and Pandeiros Rivers and (b) Tombadouro
                                                              a
      Creek and Jacar´ River arranged from chromosomes probed with 5SHind III-DNA counterstained with
                     e
      propidium iodide. Bar = 5 μm.



                                                 DISCUSSION

K A RY O T Y P I C D ATA
Karyomorph 2n = 40F
   In addition to specimens from Tombadouro Creek, all samples from the S˜ o Fran-
                                                                             a
cisco drainage shared the same karyomorphic formula, which is considered to be
characteristic for S˜ o Francisco basin populations (Bertollo et al., 2000). The dis-
                     a
tributional pattern of heterochromatin was similar to that described by Dergam &
Bertollo (1990) for the Trˆ s Marias population in this basin (Fig. 1), except for the
                            e
presence of a heterochromatic block that was always restricted to one homologue
of the first chromosome pair in males. This pattern suggested a probable XX/XY
sex chromosome system in these populations, because this variant occurred only
in males and was absent in females. This sex chromosome differentiation involv-
ing only heterochromatinization is a novelty within H. malabaricus, because all
other sex chromosome systems described for this species complex involve transloca-
tions between chromosome pairs (e.g. karyomorphs 2n = 39/40D and 2n = 40/41G)
(Bertollo et al., 2000), or translocation and heterochromatinization, as proposed for
the evolution of the 2n = 42B karyomorph from a 2n = 42A karyomorph ancestor

                                                                                                       © 2009 The Authors
       Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
PHYLOGEOGRAPHY OF HOPLIAS MALABARICUS                                                       2335




                                                                            40F Pará River




                                                                            40F Pandeiros River

                                                                            Ibimirim
                                                                                                            Haplogroup II A
                                                                            40F Pandeiros River
                                                                             40F Itapicuru River
                                                                             40F Curvelo River

                                          100, 1·00, 100, 100               Ibimirim

                                                                           40F Pandeiros River
                                                                           40F Pará River

                                                                             40F Curvelo

                                                                    40F Pandeiros River
       66, 1·00, 83, 71                                     42B Carioca Lake
                                                           42A São Mateys River
                                                           42B Dom Helvécio Lake
                                                               42 A São Mateus River
                                                             42A São Mateus River
                                100, 1·00, 98, 98         42B Dom Helvécio Lake
                                                           42A São Mateus River
                                                                         42A Macaé River
                                                63, 1·00, 86, 64            42A São João River
                  62, 0·97, *, 61                                                                           Haplogroup II B
                                                                                42 A Paraíba do Sul River
                                                                      42A Itabapoana River
                                               99, 1·00, 90, 100
                                                                      42A Paraiba do Sul River

                                                                      42A Tombadouro Creek




                                                                      42A Jacaré River




                                                         Macacu
                          100, 1·00, 83, 100            Paranaguá
                                                       42A Ribeira River                                    Haplogroup I
                                                            42A Tombadouro Creek
                                                                                 Hoplias lacerdae
                0·02

Fig. 6. Phylogenetic relationships of ATPase 6 haplotypes derived from Hoplias malabaricus karyomorphs.
      The topology was obtained with neighbour joining analysis. Bootstrap values are expressed as neighbour
      joining–Bayesian–maximum likelihood–maximum parsimony. The asterisk indicates a polytomy with
      maximum likelihood analysis. Bar = molecular distance.




© 2009 The Authors
Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
2336                                             U. SANTOS ET AL.


(Born & Bertollo, 2000). The 2n = 40/41G karyomorph also has a large first pair of
metacentrics and is therefore morphologically closest to the 2n = 40F karyomorph.
Females with both karyomorphs have identical karyotypes and may be sister kary-
omorphs or species. Ongoing studies including these karyomorphs, using additional
chromosomal markers will allow for a more thorough hypothesis of the evolutionary
origins of this sex chromosome system.
   C-positive heterochromatic bands were always located in the centromeric/
pericentromeric region of all chromosomes and in the telomeric region of some
pairs, in addition to multiple Ag-NORs sites. These heterochromatic and NOR char-
acteristics were similar to those reported for other populations or karyomorphs of
H. malabaricus (Dergam & Bertollo, 1990; Haaf et al., 1993; Bertollo, 1996, 1997;
Born & Bertollo, 2000; Vicari et al., 2003, 2005). In the Pandeiros River sample,
Ag-NORs were restricted to telomeres of some chromosomes and appeared in larger
numbers than 18S rDNA sites, but the latter also hybridized to the pericentromeric
region of one chromosome pair [Fig. 3(a)]. There is currently no explanation for the
apparently non-specific nature of some telomeric Ag-NORs in the Pandeiros sample,
which is as large as seven in the Trˆ s Marias region (Dergam & Bertollo, 1990). On
                                    e
the other hand, pericentromeric 18S rDNA sites were not visualized by the silver
nitrate method (these data and Dergam & Bertollo, 1990), which may be due to
preferential telomeric NOR activation, as already pointed out for other Hoplias spp.
karyomorphs (Vicari et al., 2005). Hybridization sites of the 5S rDNA probe were
interstitially located in a small metacentric pair in both populations, a pattern also
reported for H. malabaricus samples from Trˆ s Marias (Ferreira et al., 2007). The
                                                e
patterns obtained with repetitive DNA class 5SHind III-DNA were similar to those
obtained for the Trˆ s Marias population (Ferreira et al., 2007).
                     e

Karyomorph 2n = 42A
   The karyotypes of specimens from Tombadouro Creek and Jacar´ River were char-
                                                                   e
acterized as karyomorph 2n = 42A, a widespread karyotypic form in the Neotropics
(Bertollo et al., 2000). C-banding slowed conspicuous centromeric blocks in almost
all chromosomes, an overall pattern also reported by Born & Bertollo (2001) for other
Grande River 2n = 42A populations. High levels of Ag-NOR variation observed in
the Tombadouro Creek population have also been reported elsewhere in the Grande
River basin (Born & Bertollo, 2001). Jacar´ River H. malabaricus showed Ag-NORs
                                           e
restricted to one chromosome pair, characterizing the lowest number of activated
NORs reported so far as a fixed character state for any H. malabaricus population.
FISH with an 18S rDNA probe, however, showed a larger number of NORs cistrons
in both 2n = 42A karyomorph populations.
   On the other hand, FISH mapping with a 5S rDNA probe revealed differences
between the Tombadouro Creek and the Jacar´ River populations, where they
                                                    e
hybridized on a large submetacentric and a small metacentric pair, respectively.
This difference contrasted with previous reports of other H. malabaricus popula-
tions from the upper Paran´ Basin, the Aragu´ River, where both pairs showed
                             a                    a
fluorescence with this probe (Martins et al., 2006). Considering that at least some
specimens from both basins shared identical haplotypes (see below), these population
differences highlighted the great potential of this probe for population studies.
   Patterns of variation of 18S rDNA were substantially different from the ones
reported by Cioffi et al. (2009). These authors indicated the presence of five sites in

                                                                                                       © 2009 The Authors
       Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
PHYLOGEOGRAPHY OF HOPLIAS MALABARICUS                                                         2337

the 2n = 42B karyomorph and four fluorescent sites in a 2n = 42A karyomorph from
the upper Paran´ River basin, whereas the results of this study showed that the same
                 a
karyomorph in the Jacar´ and Tombadouro populations had three fluorescent sites.
                          e
This reduction in numbers was also evident in the 2n = 40F karyomorph, when
compared with the 2n = 40C and 2n = 39/40D karyomorphs, which showed five
fluorescent sites. Thus, this probe appears to be especially informative for studying
among-karyomorph variation.
   According to the haplotype-based trees, 5SHind III-DNA patterns reflected a small
increase in site numbers (from 18 to 20); Cioffi et al. (2009) also found variation
in site numbers for other 2n = 42 and 2n = 40 populations. Also, the fluorescent
centromeric region in the first or second chromosome pairs appears to be particularly
informative. The presence of the fluorescent region in the first chromosome pair was
observed in the 2n = 42A karyomorph, as is the case for 2n = 42B, 2n = 40C and
2n = 39/40D karyomorphs (Cioffi et al., 2009), representing a plesiomorphic state.
On the other hand, the 2n = 40F karyomorph showed an apparently apomorphic
condition, where the fluorescent region occurred in the second chromosome pair.
Absence of the fluorescent site in the first chromosome pair might be attributed
to a more recent origin of the first chromosome pair and its possible origin from
chromosomes lacking this site. This hypothesis, however, involves a particularly
complex evolutionary process, because the persistence of diploid number would also
involve alterations of other chromosome pairs. Alternatively, the 2n = 40F kary-
omorph might have evolved directly from a 2n = 42A ancestor, causing a reduction
in diploid number and producing an independent 2n = 40 lineage. This hypothesis is
consistent with the hypothesis of Bertollo et al. (2000), based on gross chromosome
morphology, that the 2n = 40F, 2n = 40/41G and 2n = 42E karyomorphs represent
a monophyletic group within H. malabaricus.
   In the submetacentric chromosome group, the pattern of fluorescent centromeric
sites in three major chromosome pairs also sets apart the 2n = 40F karyomorph from
other karyomorphs. The 2n = 42A karyomorph and the three karyomorphs reported
by Cioffi et al. (2009) have conspicuous fluorescent centromeric regions in these
pairs, while this number was reduced to two pairs in the 2n = 40F karyomorph.
Homeologies among other chromosome pairs seem less reliable and must await
further data. These FISH results and the presence of a unique sex chromosome
system clearly indicate that the 2n = 40F karyomorph is a taxon with the largest suit
of derived karyotypic characters in this species complex.

M O L E C U L A R D ATA
   Molecular data are congruent with the cytogenetic results and suggest a long
reproductive isolation between the karyomorphs. Haplogroup I included some of
the 2n = 42A karyomorph haplotypes from the Tombadouro Creek and haplotypes
derived from three coastal basins located south of the Para´ba do Sul River. This
                                                             ı
haplogroup showed low levels of within variation, suggesting that these haplotypes
were closely related. Some haplogroup II haplotypes were sympatric with haplogroup
I haplotypes at the Tombadouro Creek. Within haplogroup IIB, all haplotypes from
the Grande River were closely related to haplotypes from four coastal basins. These
haplotypes showed a sister group relationship with haplotypes from the S˜ o Mateus
                                                                          a
and Doce coastal basins that were not represented in samples from the S˜ o Francisco
                                                                       a

© 2009 The Authors
Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
2338                                             U. SANTOS ET AL.


and Grande Rivers. Some 2n = 42 trahiras from the Tombadouro Creek and Jacar´        e
River shared identical haplotypes, suggesting a recent range expansion from the
Grande to the S˜ o Francisco basins.
                 a
   These results provide insights into the evolution of H. malabaricus karyomorphs
and the history of contact between coastal and continental hydrological basins.
The most inclusive study of ND2 and ATPase 6 divergence between freshwater
fishes is Bermingham et al. (1997), who estimated a rate of divergence for this
gene segment of 1·3% per million years. Overall sequence (p-distance) divergence
of the divergent haplogroup I haplotypes versus haplogroup II haplotypes would
suggest a lineage split at 4·2 million years ago, an age that is younger than the
inferred Amazon–Paranean vicariance, which has been dated at 11·8–10 million bp
(reviewed in Lundberg et al., 1998). This isolation left 2n = 42 and 2n = 40 kary-
omorphs in both basins (Bertollo et al., 2000), indicating that karyotypic evolution
was well underway during the Late Miocene Epoch. Miocene fossils are also consid-
ered to be similar to present-day taxa (Lundberg, 1998). The information content of
molecular variation of this species complex may be more elusive: a preliminary
analysis of ATPase 6 sequences in H. malabaricus populations from the Ama-
zon–Paranean boundary basins reveals two clades that show large differences in
p-distance divergence within each clade (0·10 and 0·34% per million years, respec-
tively) (J. A. Dergam, unpubl. data), suggesting that molecular evolution within the
H. malabaricus complex may be highly variable and influenced by other causes. Fac-
tors such as efficiency in DNA repair mechanism (Britten, 1986), generation time
(Wu & Li, 1985), metabolic rate (Martin & Palumbi, 1993) and demographic pro-
cesses (Ohta, 2002) may affect substitution rates among taxa. Hence, it is plausible
to hypothesize a strong effect of demographic processes on the rates of molecular
substitution in these populations, considering the close phylogenetic relationships
among H. malabaricus karyomorphs and the adaptation of H. malabaricus to life in
small populations, which is also reflected in their high levels of karyotypical vari-
ation. Slightly disadvantageous substitutions can drift to high frequencies in small
populations (Ohta, 2002), thereby altering substitution rates in the DNA. Therefore,
H. malabaricus and other species with similar ecology may be particularly poor can-
didates for using the molecular clock hypothesis to estimate divergence times. In the
highly migratory species, Prochilodus spp., substitution rates of ATPase 6,8 range
from 0·8 to 2·5% (Sivasundar et al., 2001), suggesting haplotypes in H. malabaricus
are among the most divergent in the Neotropical region. The present study is the first
to indicate such a deep molecular divergence within the same karyomorph, although
deep cytochrome b divergences were also observed among populations of the catfish
Pimelodus albicans (Valenciennes) in the River Plate basin (Vergara et al., 2008).
   The location where lineage splitting occurred is limited to the distribution of
current karyomorphs. The Paran´ River basin harbours at least three karyomorphs,
                                  a
whereas the 2n = 40F karyomorph appears to be widespread in the S˜ o Francisco
                                                                          a
River basin and the 2n = 42A karyomorph is apparently restricted to the Par´ River
                                                                                a
headwaters. North-eastern coastal populations harbour the 2n = 40F karyomorph
as far as the Buranh´ m River to the south (J. A. Dergam, unpubl. data) (Fig. 1),
                      e
and these haplotypes had the lowest degree of divergence. This macro-geographical
pattern suggests that lineage splitting occurred elsewhere and not in the relatively
isolated S˜ o Francisco River basin. In haplogroup IIB, the close relationship between
           a
the 2n = 42A and 2n = 42B karyomorphs indicated that karyotypical differentiation

                                                                                                       © 2009 The Authors
       Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
PHYLOGEOGRAPHY OF HOPLIAS MALABARICUS                                                         2339

involved few alterations that resulted in a differentiated XX/XY chromosome system
(Bertollo et al., 2000).

S T R E A M P I R AC Y
   On a local scale, the apparently restricted range of the Tombadouro Creek 2n =
42A karyomorph may have resulted from stream piracy from a former Jacar´ River  e
tributary. The Tombadouro Creek is 12 km from the Jacar´ across the Galga water-
                                                              e
shed. Three hypotheses might explain this dispersal. First, boundary crossing may
have resulted from either human mediated activities. Second, dispersal may have
occurred through a high-altitude wetland that used to drain into both basins. For
example, the connection may have been destroyed by the construction of BR 494
Highway, which is 8 km away at its closest point from the Tombadouro Creek. Third,
headwater capture in the region may have occurred by relatively recent reactivation
of ancient faults (Saadi et al., 2002). Altitudinal characteristics are also consistent
with the direction of stream capture. Tombadouro is at 860 m altitude, whereas the
Jacar´ River is at 1038 m. This area is within the range of a continental fault known
      e
as the Upper Grande River Crustal Discontinuity (Saadi et al., 2002). This fault
represents the southern limit of the S˜ o Francisco craton and the movable belts of
                                        a
southern Minas Gerais State (Fig. 1) and has been active in the last 15 000 years.
   The phylogenetic origins of two highly divergent haplotypes in the Tombadouro
Creek became clear only after haplotypes from coastal basins were included in the
analysis. Most critically, haplotypes found in sympatry in the S˜ o Francisco River
                                                                     a
are related to haplotypes in different coastal basins. Under the allopatric model of
speciation, this asymmetry suggests that ancestral fish dispersed from the coastal to
the continental drainages, and the present study is the first to indicate unambiguously
the direction of this dispersal. The timing of the dispersal between coastal and inland
drainages, however, could not be estimated, because molecular clock expectations are
not suitable for H. malabaricus. Therefore, the phylogeographic history of this group
does not fit any of the three phylogenetic patterns proposed by Ribeiro (2006). On the
other hand, low genetic divergence between the S˜ o Francisco and Itapicuru River
                                                      a
drainages may be part of a recent faunal exchange in the arid region of north-eastern
Brazil that is apparent in many other species of fish (Rosa et al., 2004).
   Ribeiro (2006) indicated that the Atlantic drainages (Doce, Para´ba do Sul and
                                                                         ı
Ribeira) have existed since the break-up of Gondwana. The results of the present
study suggested that populations in these basins represent three biogeographic units,
for which only two have close representatives in the boundaries of the Jacar´ and Par´
                                                                             e        a
drainages. Dergam et al. (2002) previously reported a close phylogenetic relatedness
between two haplotypes in the Doce and lower Grande River basins. Fish dispersal
from coastal to continental drainages may have been restricted to only a few taxa
adapted to headwater conditions, because coastal drainages are characterized by high
levels of endemism (Ribeiro, 2006) and the Proterozoic Mantiqueira Range is con-
sidered to be an efficient barrier between coastal and continental drainages (Ingenito
& Buckup, 2007). To the south, stream piracy has been particularly intensive in the
Para´ba do Sul, Ribeira and Upper Paran´ basins (Ribeiro, 2006).
     ı                                     a
   The present study provided important information on the population distribution
of H. malabaricus in S˜ o Francisco River basin and on historical relationships with
                         a
populations in the Grande River and coastal basins. The 2n = 40F karyomorph and

© 2009 The Authors
Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
2340                                             U. SANTOS ET AL.


its associated haplotypes were widely distributed throughout the S˜ o Francisco basin,
                                                                     a
maintaining an apparently stable karyotypic structure of the chromosomal markers
analysed here. This karyomorph was characterized by a XX/XY sex chromosome
system. In contrast, the 2n = 42A karyomorph showed a more restricted distribution
in S˜ o Francisco basin and was closely associated with populations in a Grande
     a
River basin tributary, where the 2n = 42A karyomorph predominated. The 2n = 42A
karyomorph populations isolated in drainages, however, showed striking differences
for some markers, notably the Ag-NORs and 5S rDNA sites. The patterns of variation
in repetitive DNA sequences as revealed by 5S rDNA probes indicated that these
markers appeared to be useful population markers, showing significant differences
among localities and karyomorphs. Patterns of 5SHind III-DNA variation yielded the
largest phylogenetic signal and placed the 2n = 40F karyomorph in a high position
in the phylogeny of karyomorphs that occur in eastern Brazil.
   Given the current status of phylogenetic data, an understanding of distribution
patterns for H. malabaricus will be particularly challenging. Studies of the distri-
bution patterns of freshwater fishes have considered endemism (Vari, 1988), faunal
composition including endemic and widespread species (Hubert & Renno, 2006),
molecular data (Sivasundar et al., 2001; Montoya-Burgos, 2003) or a combination
of morphological and molecular data (Menezes et al., 2008). When integrated into a
wider geographic context, molecular and cytogenetic patterns can be informative of
faunal dispersals between drainages. The results of the present study suggested that
recent fish dispersal may occur even within apparently stable geological regions and
that headwater stream piracy between coastal and continental (upper Paran´ tribu-
                                                                                a
taries) basins may have played a role in producing the high levels of freshwater fish
diversity in the Neotropics.
   Finally, the topology of mtDNA-based tree suggests that the proposal of Dergam &
Bertollo (1990) to separate species by diploid numbers would result in a paraphyletic
arrangement, in which some Hoplias spp. haplotypes associated with 2n = 42 kary-
omorphs would be more closely related to trahiras with 2n = 40 karyomorphs than
to other fish with 2n = 42 karyomorphs. Although haplogroup I appeared as the sis-
ter group of the remaining trahiras, haplogroup II is less supported and the possible
ancestry of karyomorphs must wait for more data. A time frame for dispersal between
coastal and continental drainages is elusive for the H. malabaricus species complex.
Nevertheless, geographical isolation during coastal diversification has enhanced spe-
ciation processes and genetic isolation between karyomorphs, a process that has
resulted in molecular and cytogenetic differentiation between populations. These
results are consistent with the existence of multiple independent lineages that can be
easily detected with standard cytogenetic techniques.
   Although Hoplias spp. are common members of lowland freshwater communities
(Bonetto et al., 1969; Winemiller, 1991), at least part of their widespread distribution
in the Neotropics is due to their ability to colonise high-altitude headwaters, such
as in the Jacar´ and Tombadouro localities. The question of how this non-migratory
                e
sedentary species has adapted to sluggish waters and to waters at high altitudes is not
explained by current geological data. Nevertheless, this broad ecological diversity
indicates a long history of Hoplias spp. in south-eastern Brazil.

  J. T. da Costa, J. B. Santos, R. de F. Guimar˜ es and A. M. R. de Souza provided support
                                               a
during fieldwork. W. Evangelista and U. Jacobina collected samples from the Ibimirim lakes

                                                                                                       © 2009 The Authors
       Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
PHYLOGEOGRAPHY OF HOPLIAS MALABARICUS                                                         2341

and Itapicuru River, and T. L. Pereira assisted with some laboratory work. M. Wachlevski
supported the initial stage of this study. The authors wish to thank two anonymous review-
ers for their suggestions. Financial support for this study was provided by the Program for
the Advancement of Research of the Universidade do Estado de Minas Gerais, the National
Council for Technological and Scientific Development of the Brazilian Government, the Uni-
versidade Federal de Vicosa and the Fundacao Educacional de Divin´ polis-FUNEDI/UEMG.
                         ¸                  ¸˜                       o


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© 2009 The Authors
Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343

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Molecular and cytogenetic phylogeography of h. malabaricus

  • 1. Journal of Fish Biology (2009) 75, 2326–2343 doi:10.1111/j.1095-8649.2009.02489.x, available online at www.interscience.wiley.com Molecular and karyotypic phylogeography in the Neotropical Hoplias malabaricus (Erythrinidae) fish in eastern Brazil ¨ U. Santos*, C. M. Volcker†, F. A. Belei*, M. B. Cioffi‡, L. A. C. Bertollo‡, S. R. Paiva§ and J. A. Dergam* *Laborat´ rio de Sistem´ tica Molecular Beagle, Departamento de Biologia Animal, o a Universidade Federal de Vi¸ osa, 36570-000 Vi¸ osa, MG, Brazil, †Rua Edgard Coutens de c c Menezes, 47. 28970-000 Pontinha, Araruama, RJ, Brazil, ‡Centro de Ciˆ ncias Biol´ gicas e o e da Sa´ de, Departamento de Gen´ tica e Evolu¸ ao, Universidade Federal de S˜ o Carlos, u e c˜ a S˜ o Carlos, SP, Brazil and §Embrapa Recursos Gen´ ticos e Biotecnologia, Laborat´ rio a e o Gen´ tica Animal, PCG, Parque Esta¸ ao Biol´ gica, Final Av. W/5 Norte, Bras´lia, DF, Brazil e c˜ o ı (Received 9 June 2009, Accepted 13 October 2009) The sedentary, predatory characin Hoplias malabaricus has one of the widest distributions of fresh- water fishes in South America and is characterized by seven karyomorphs (A–G) that occur in sympatric and allopatric populations. Karyotypical patterns of variation in wild populations have been interpreted as evidence of multiple lineages within this nominal species, a possibility that may limit the validity of experimental data for particular karyomorphs. This study used the phylogeo- graphic and genealogical concordance between cytogenetic (N = 49) and molecular (mitochondrial DNA) (N = 73) data on 17 samples, collected in 12 basins from south-eastern and north-eastern Brazil, to assess the systematic value of cytogenetic data. Cytogenetic patterns show a sex chro- mosome system in the 2n = 40F karyomorph. Molecular and cytogenetic data indicate a long, independent evolutionary history of karyomorphs and a coastal origin of continental populations in south-eastern Brazil. The lack of fit with molecular clock expectations of divergence between groups is likely to be due to strong demographic fluctuations during the evolution of this species complex. The results indicate that karyotypical identification provides a reliable baseline for placing experimental studies on Hoplias spp. in a phylogenetic context. © 2009 The Authors Journal compilation © 2009 The Fisheries Society of the British Isles Key words: freshwater fish; in situ hybridization; molecular clock; mtDNA; repetitive DNAs; South America. INTRODUCTION The Neotropical region harbours at least 6025 species of freshwater fishes (Reis et al., 2003), but perhaps as many as 8000 (Schaefer, 1998), the result of a long evo- lutionary history of isolation and specialization, involving mainly otophysan fishes. Freshwater fishes are highly suitable for the recovery of past biogeographic processes, due to their obligatory relationship with water (Myers, 1938). The biogeographical Author to whom correspondence should be addressed. Tel.: +55 31 3899 2555; fax: +55 31 3899 2578; email: dergam@ufv.br 2326 © 2009 The Authors Journal compilation © 2009 The Fisheries Society of the British Isles
  • 2. PHYLOGEOGRAPHY OF HOPLIAS MALABARICUS 2327 information relevant for fish diversification has been reviewed by Lundberg et al. (1998) and Ribeiro (2006). The former revision focused on the evolution of Andean geomorphology and the effects of this process on the South American continent, whereas the latter work dealt with the evolution of particular coastal drainages, their geomorphological interactions with continental basins and the expected lev- els of phylogenetic divergence among fishes derived during these complex temporal events. Among Neotropical freshwater fishes, the trahira, Hoplias malabaricus (Bloch), has one of the largest distributional ranges, occurring from Panam´ to the Buenos a Aires Province in Argentina (Berra, 2007). H. malabaricus is well adapted to life in small, isolated populations, in conditions that may facilitate the stochastic fixa- tion of chromosomal rearrangements (Sites & Moritz, 1987). This species is one of the cytogenetically most studied taxa and shows a conspicuous karyotypic diversi- fication, with up to seven karyomorphs with diploid numbers ranging from 39 to 42. Three groups of karyomorphs are readily evident, based on distribution ranges. Two karyomorphs, 2n = 42A and 2n = 40C, are the most widespread and occur in sympatry in the lower Paran´ –Paraguay and Amazon River basins. A second, less a widely spread group is represented by three karyomorphs: (1) 2n = 40F occurs in the S˜ o Francisco River basin, in the lower portions of the Tocantins River, in coastal a drainages in Surinam and in minor basins in north-eastern Brazil; (2) 2n = 39/40D occurs in the upper Paran´ River (in sympatry with 2n = 42A) and (3) 2n = 40/41G a occurs exclusively in the Amazonian Basin in the Aripuan˜ , Madeira and Trombetas a Rivers. The third group consists of karyomorphs with much restricted distributions. For example, 2n = 42B occurs in the Doce River basin (also in sympatry with 2n = 42A), and 2n = 42E occurs only in the Trombetas River (Bertollo et al., 2000). The lack of hybrids between sympatric karyomorphs has been interpreted as evi- dence for the existence of several distinct species within this nominal taxon (Bertollo et al., 1986, 2000). Therefore, uniparental molecular markers can be a particularly useful complement to assess gene flow in wild populations. To date, molecular stud- ies on H. malabaricus have been restricted to populations in the Doce River basin (Dergam et al., 2002) and the Iguacu River basin (Dergam et al., 1998). Both studies ¸ indicated phylogenetically related populations in the coastal and continental basins, but failed to reveal the direction of dispersal events. Karyotypic and molecular patterns of variation may reveal vicariances and dis- persals that provide insights into evolutionary processes that may involve other components of the aquatic fauna. The congruence of independent molecular and cytogenetic characters may give support to the hypothesis that at least some kary- omorphs behave as valid species within H. malabaricus. A better understanding of H. malabaricus species-level systematics is needed to interpret correctly the compar- ative physiological, behavioural and anatomical studies in an explicit phylogenetic framework (Harvey & Pagel, 1991). Biodiversity patterns in this species complex also bear potential information to understand the palaeohydrology of the continent. In the present study, patterns of cytogenetic and molecular variation of H. malabaricus populations in the S˜ o Francisco, Grande and in several coastal basins along eastern a Brazil were analysed using molecular and cytogenetic techniques. © 2009 The Authors Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
  • 3. 2328 U. SANTOS ET AL. MATERIALS AND METHODS S P E C I M E N S A N D C H R O M O S O M E P R E PA R AT I O N Cytogenetic analyses were carried out on 49 specimens collected in the Pandeiros River, Par´ River and its headwaters in the Tombadouro Creek and in the Jacar´ River (Table I a e and Fig. 1). Cell division was stimulated in vivo with two daily applications of Munolan, a commercially available antigen lysate, following Molina (2001). Mitotic chromosomes were obtained from cell suspensions of the anterior kidney, using the conventional air-drying method (Bertollo et al., 1978). Fish were previously anaesthetized with clove oil (Henyey et al., 2002). Secondary data from 10 populations with known chromosome numbers were also included in the analysis. C H R O M O S O M E S TA I N I N G In addition to the standard Giemsa method, chromosomes were analysed using silver nitrate staining (Howell & Black, 1980) to visualize the nucleolar organizing regions (Ag-NOR). C-banding was also employed to detect C-positive heterochromatin (Sumner, 1972). (4 ,6 diamidino-2-phenylindole dihydrochloride (DAPI) and chromomycin A3 (CMA3 ) fluores- cence staining were used to identify the chromosome adenine–thymine (AT) and guanine- cytosine (GC) rich regions, respectively (Sola et al., 1992). Some populations with unknown karyotypes (or whose karyotypes are being studied by J.A.D.) were also included in this study (see below). C H R O M O S O M E H Y B R I D I Z AT I O N A N D K A RY O T Y P I C A N A LY S I S Fluorescent in situ hybridization (FISH) was performed according to Pinkel et al. (1986) using three repetitive DNA sequences probes isolated from the genome of H. malabaricus and an 18S probe. The first probe contained a 5S rDNA repeat copy and included 120 base pairs Table I. Hoplias malabaricus collecting localities, sample sizes, geographic coordinates and karyomorph nomenclature Cytogenetic Molecular Locality samples samples G.P.S. Karyomorph Par´ River a 8♀ –7♂ 14 20◦ 08 21 S–44◦ 53 17 W 40F Pandeiros River 2♀ –3♂ 9 15◦ 40 18 S–44◦ 37 43 W 40F Tombadouro Creek 4♀ –3♂ 5 20◦ 41 56 S–44◦ 34 46 W 42A Ibimirim — 6 8◦ 30 31 S–37◦ 42 21 W Unknown Curvelo 12 8 18◦ 42 57 S–44◦ 25 56 W 40F Itapicuru River 4 1 13◦ 14 40 S–41◦ 23 34 W 40F Dom Helv´ cio Lake e 10 3 19◦ 46 34 S–42◦ 35 19 W 42B S˜ o Mateus River a 2 6 18◦ 44 09 S–48◦ 29 47 W 42A Para´ba do Sul River ı 15 2 21◦ 46 26 S–41◦ 30 01 W 42A Itabapoana River 8 2 21◦ 08 20 S–41◦ 39 35 W 42A Maca´ River e 6 1 22◦ 17 43 S–41◦ 52 48 W 42A S˜ o Jo˜ o River a a 8 2 22◦ 33 29 S–42◦ 07 21 W 42A Ribeira River 2 1 24◦ 29 23 S–47◦ 50 10 W 42A Carioca Lake 15 1 19◦ 45 32 S–42◦ 37 15 W 42B Paranagu´ Bay a — 1 25◦ 32 39 S–48◦ 29 47 W Unknown Macacu Waterfalls — 1 22◦ 27 37 S–42◦ 39 18 W Unknown Jacar´ River e 15♀ –7♂ 10 20◦ 48 29 S–44◦ 33 58 W 42A © 2009 The Authors Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
  • 4. PHYLOGEOGRAPHY OF HOPLIAS MALABARICUS 2329 Equator 35° 44′ 58′′ W 8° 08′ 01′′ S 1 500 km 1 - Ibimirim 2 2 - Itapicuru River 3 - Pandeiros River 3 4 - Buranhém River 5 - Curvelo 6 - Três Marias 7 - São Mateus River 4 8 - Dom Helvécio and Carioca lakes 5 9 - Pará River 6 7 10 - Tombadouro Creek 8 9 11 - Itabapoana 10 12 11 12 - Jacaré River 13 13 - Paraíba do Sul River 14 14 - Macacu 16 15 15 - Macaé 17 18 16 - São João River 26° 19′ 17′′ S 17 - Ribeira River 54° 35′ 13′′ W 18 - Paranaguá Fig. 1. Hoplias malabaricus collection localities. The dotted line indicates the upper Grande crustal disconti- nuity. The locality of Trˆ s Marias is also depicted. e (bp) of the 5S rRNA encoding gene and 200 bp of the non-transcribed spacer (NTS) (Martins et al., 2006). The second probe is specific to H. malabaricus (Ferreira et al., 2007) and contained a copy of the repetitive satellite 5SHind III-DNA sequence with 360 bp composed of a 95 bp segment similar to the 5S rRNA gene of the first probe and a 265 bp segment similar to the NTS of the first probe (Martins et al., 2006). The third probe corresponded to a 1400 bp segment of the 18S rRNA gene obtained by the polymerase chain reaction (PCR) from nuclear DNA (Cioffi et al., 2009). The probes were labelled by nick translation with biotin-14- dATP (Bionick Labeling System, Invitrogen; www.invitrogen.com). Signal detection and its amplification were performed using conjugated avidin–fluorescein isothiocyanate (FITC) and anti-avidin–biotin (Sigma; www.sigmaaldrich.com). The chromosomes were counterstained with propidium iodide (50 μg ml−1 ) and analysed with an Olympus BX50 epifluorescence microscope. The chromosomal images were captured using CoolSNAP-Pro software (Media Cybernetics; www.MediaCy.com). About 30 metaphase spreads were analysed per specimen to determine the diploid chromosome number and karyotypic structure. Chromosomes were classified as metacentrics (m) or submetacentrics (sm), according to centromeric index values proposed by Levan et al. (1964). D N A E X T R A C T I O N A N D M O L E C U L A R A N A LY S I S DNA extraction followed Boyce et al. (1989). Fragments of ATPase 6 were amplified using primers L8524 (5 -AAY CCT GAR ACT GAC CAT G-3 ) and H9236 (5 -GTT AGT GGT CAK GGG CTT GGR TC-3 ) (Quenouille et al., 2004). Double-stranded DNA was syn- thesized in 50 μl reactions containing 10 μl dNTPs (1 mM), 5 μl reaction buffer (200 mM Tris–HCl pH 8·4, 500 mM KCl), 2 μl MgCl2 (50 mM), 2 μl of each primer (10 mM), 0·5 μl (2·5 U) Taq DNA polymerase (Phoneutria), 2 μl template DNA (100 ng/μl) and 26·5 μl H2 O. PCR conditions were as follows: 94◦ C (2 min), five cycles of 94◦ C (45 s), 54◦ C (45 s) and 72◦ C (1·5 min) and 29 cycles of 94◦ C (45 s), 58◦ C (45 s) and 72◦ C (1·5 min). PCR products were purified using Qiaquick (Qiagen; www.quiagen.com): 5 μl of the purified PCR product were used in a 10 μl cycle sequencing reaction using a drhodamine terminator © 2009 The Authors Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
  • 5. 2330 U. SANTOS ET AL. cycle sequencing kit (PE Applied Biosystems; www.appliedbiosystems.com). Sequences were aligned using CLUSTAL X 1.83 in MEGA 4.0 (Tamura et al., 2007). Phylogenetic trees were constructed with neighbour joining (NJ) (Saitou & Nei, 1987), maximum parsimony (MP), maximum likelihood (ML) (Felsenstein, 1981) and Bayesian inference (MB) (Huelsenbeck & Ronquist, 2001). The model of molecular evolution that best fitted the data was chosen using Modeltest 3.7 (Posada & Crandall, 1998), and haplotype divergence was estimated within and between haplogroups using this selected model. Phylogenetic signal in NJ, MP and ML trees was assessed using bootstraps with 1000 repetitions. Bayesian inference was performed with five million Markov-Chain Monte-Carlo (MCMC) steps to produce posterior probabil- ities of nodes in the tree with MrModeltest 2 (Nylander, 2004). Hoplias lacerdae Miranda Ribeiro was used as an outgroup. Sequences were deposited in GenBank (accession numbers GQ848606–GQ848642). RESULTS K A RY O T Y P I C D ATA Karyomorph 2n = 40F All specimens from the Pandeiros and Par´ rivers (except those from Par´ ’s a a headwaters, in Tombadouro Creek) had 2n = 40 chromosomes for both sexes. The karyotypes were composed of 10 m + 10 sm pairs, without morphologically differ- entiated sex chromosomes using Giemsa conventional staining [Fig. 2(a)], and were typical 2n = 40F karyomorphs. C-banding showed heterochromatic blocks in cen- tromeric regions, except for chromosome pairs 9 and 10, which had less conspicuous blocks, in addition to faint telomeric marks in some chromosome pairs. Males always showed an interstitial heterochromatic block in the short arms of one homologue of the first chromosome pair [Fig. 2(c)], whereas females lacked this block [Fig. 2(e)]. This heterochromatic region was negative for DAPI staining but failed to show fluorescence with CMA3 . A proximal heterochromatic block located on the short arms of a small submetacentric pair was the only observed GC-rich segment in both populations [Fig. 3(f), (i)]. Ag-NORs were telomeric and their numbers were either fixed (four in the Par´ River) or varied from 4 to 5 (Pandeiros River) [Fig. 4(a), (b)]. a FISH analyses with the repetitive sequences showed no differences between these two populations. The 18S rDNA probe hybridized on the pericentromeric region of one metacentric pair and on the telomeric regions of two submetacentric pairs [Fig. 3(a)]. The 5S rDNA probe hybridized on the pericentromeric region of a medium-sized metacentric pair [Fig. 3(c)]. The repetitive 5SHind III-DNA was mapped in the cen- tromeric region of 10 chromosome pairs [Fig. 5(a)]. Although Ibimirim samples were not karyotyped, they were collected in a region with populations characterized by 2n = 40F karyomorph and were assumed to share this karyomorph. Karyomorph 2n = 42A In the headwaters of Tombadouro Creek, all specimens had 2n = 42 chromosomes, with a karyotype consisting of 11 m + 10 sm pairs and without morphologically dif- ferentiated sex chromosomes [Fig. 2(b)], and were considered to be typical 2n = 42A karyomorphs. Besides some telomeric marks, conspicuous heterochromatic bands were found in the centromeric region of all chromosomes, except for chromosome pairs 6, 19 and 20, which showed faint blocks [Fig. 2(d)]. Ag-NORs varied among and within individuals (one to two pairs), and bitelomeric NORs were evident in either one or two chromosomes [Fig. 4(c)]. In this population, the 18S rDNA probe © 2009 The Authors Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
  • 6. PHYLOGEOGRAPHY OF HOPLIAS MALABARICUS 2331 (a) (b) m m 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 10 11 sm sm 11 12 13 14 15 16 17 18 19 12 13 14 15 16 17 18 19 20 20 21 (c) (d) m 1 2 3 4 5 6 7 8 9 m 1 2 3 4 5 6 7 8 9 10 10 11 sm sm 11 12 13 14 15 16 17 18 19 12 13 14 15 16 17 18 19 20 20 21 (e) (f) m m 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 10 11 sm sm 11 12 13 14 15 16 17 18 19 12 13 14 15 16 17 18 19 20 20 21 5 μm Fig. 2. Giemsa-stained and C-banded karyotypes of Hoplias malabaricus. (a) conventional Giemsa-stained karyotype of populations from Pandeiros and Par´ rivers, (b) samples from Jacar´ River and Tombadouro a e Creek, (c) C-banded karyotypes of males from Par´ and Pandeiros Rivers, (e) C-banded karyotypes of a females from Pandeiros and Par´ Rivers, (d) C-banded karyotype of specimens from Tombadouro Creek a and (f) C-banded karyotype from Grande River. Arrows indicate an interstitial heterochromatic block in the short arm of a homologue of the first chromosome pair. This block appeared in males of Pandeiros and Par´ River populations, but not in females. a hybridized on three chromosome pairs, with interstitial, telomeric and bitelomeric sites [Fig. 3(b)]. The 5S rDNA probe hybridized on the interstitial region of a large submetacentric pair [Fig. 3(d)]. The repetitive 5SHind III-DNA probe bound to the centromeric region of nine chromosome pairs [Fig. 5(b)]. Specimens from the Jacar´ River in the Grande drainage had 2n = 42 kary- e omorphs composed of 11 m + 10 sm pairs, without morphologically differentiated sex chromosomes [Fig. 2(b)], and represented a typical 2n = 42A karyomorph. Het- erochromatic blocks were centromeric in most chromosomes, except for pairs 6, 8, 11, 19 and 20, which showed faint bands, in addition to telomeric marks in some chromosome pairs [Fig. 2(f)]. Ag-NORs were restricted to telomeric regions of two chromosomes [Fig. 4(d)]. Fluorescent regions hybridized with 18S rDNA and 5SHind III-DNA and showed a pattern similar to the Tombadouro Creek sample. © 2009 The Authors Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
  • 7. 2332 U. SANTOS ET AL. (a) (b) (c) (d) (e) (f) (g) (h) (i) Fig. 3. Metaphase chromosome spreads of Hoplias malabaricus after FISH with 18S and 5S rDNA probes and DAPI–Chromomycin A3 staining. Numerical and positional variation of 18S rDNA sites in populations from (a) Pandeiros and Par´ Rivers, (b) Tombadouro Creek and Jacar´ River. Mapping of 5S rDNA sites a e for populations of (c) Pandeiros and Par´ Rivers, (d) Tombadouro Creek and (e) Jacar´ River. DAPI a e staining demonstrating absence of signals in males (f) and females (h) from Par´ River. Arrow indicates a the interstitial heterochromatic block negative for DAPI in one homologue of the first chromosome pair in males. Sequential CMA3 staining showing GC-rich DNA segments located on a submetacentric chromosome pair on (g) males and (i) females. Bar = 5 μm. Additionally, the 5S rDNA probe hybridized on the interstitial region of a small metacentric pair [Fig. 3(e)]. As was the case with the Ibimirim samples, Paranagu´ a and Macacu samples were collected within the range of 2n = 42A populations (J. A. Dergam, unpubl. data) and were therefore assumed to be derived from these populations. M O L E C U L A R D ATA Multiple sequences 511 bp in length yielded 86 phylogenetically informative sites. Translation of these sequences into the corresponding amino acid sequences resulted in eight phylogenetically informative sites. The transitions:transversions ratio was 6·4, indicating that substitution rates were not saturated. The best model fit esti- mated with Modeltest and MrModeltest was HKY+G, which was used for NJ. In the tree, haplotypes were separated into two large clades with variable boot- strap support (Fig. 6). The largest bootstrap values were obtained for haplogroup I, which was composed of the 2n = 42A karyomorph from Tombadouro Creek, Ribeira, Paranagu´ and Macacu rivers (Fig. 1). Haplogroup II was less supported and included a all remaining H. malabaricus (N = 67), regardless of diploid number. Within this © 2009 The Authors Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
  • 8. PHYLOGEOGRAPHY OF HOPLIAS MALABARICUS 2333 (a) (b) 5 μm 5 μm (c) (d) 5 μm 5 μm Fig. 4. Metaphase chromosome spreads of Hoplias malabaricus after silver staining, showing Ag-NORs in populations from (a) Par´ River, (b) Pandeiros River, (c) Tombadouro Creek and (d) Jacar´ River. a e Arrows and arrowhead indicate telomeric and bitelomeric Ag-NORs, respectively. Table II. Within- and between-haplogroup molecular distances in Hoplias malabaricus Haplogroup I, Haplogroup IIA, Haplogroup IIB, 2n = 42 2n = 40 2n = 42 Haplogroup I, 2n = 42 0·032 Haplogroup II A, 2n = 40 6·82 0·0053 Haplogroup II B, 2n = 42 9·20 9·50 2·65 haplogroup, two subclades were highly supported: haplotypes derived from popula- tions with the 2n = 40F karyomorph (haplogroup IIA) and from populations with 2n = 42A and 2n = 42B karyomorphs (haplogroup IIB). Haplogroup IIA included all 2n = 40 S˜ o Francisco River H. malabaricus, plus the Itapicuru River specimen. a Haplogroup IIB included two well-defined lineages: one included haplotypes from the Doce and S˜ o Mateus Rivers with no representatives in the Grande River, and a a second lineage indicated a close phylogenetic relatedness between H. malabaricus from the Jacar´ (Grande River), the Tombadouro (S˜ o Francisco River) and the e a Maca´ , S˜ o Jo˜ o, Itabapoana and Para´ba do Sul coastal basins. Within-haplogroup e a a ı nucleotide molecular distances showed high levels of variation, with the smallest val- ues in the S˜ o Francisco and Itapicuru samples and the largest in the IIB haplogroup. a The range of between-haplogroup variation was much smaller (Table II). © 2009 The Authors Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
  • 9. 2334 U. SANTOS ET AL. (a) m 1 2 3 4 5 6 7 8 9 10 sm 11 12 13 14 15 16 17 18 19 20 (b) m 1 2 3 4 5 6 7 8 9 10 11 sm 12 13 14 15 16 17 18 19 20 21 Fig. 5. Karyotypes of Hoplias malabaricus samples from (a) Par´ and Pandeiros Rivers and (b) Tombadouro a Creek and Jacar´ River arranged from chromosomes probed with 5SHind III-DNA counterstained with e propidium iodide. Bar = 5 μm. DISCUSSION K A RY O T Y P I C D ATA Karyomorph 2n = 40F In addition to specimens from Tombadouro Creek, all samples from the S˜ o Fran- a cisco drainage shared the same karyomorphic formula, which is considered to be characteristic for S˜ o Francisco basin populations (Bertollo et al., 2000). The dis- a tributional pattern of heterochromatin was similar to that described by Dergam & Bertollo (1990) for the Trˆ s Marias population in this basin (Fig. 1), except for the e presence of a heterochromatic block that was always restricted to one homologue of the first chromosome pair in males. This pattern suggested a probable XX/XY sex chromosome system in these populations, because this variant occurred only in males and was absent in females. This sex chromosome differentiation involv- ing only heterochromatinization is a novelty within H. malabaricus, because all other sex chromosome systems described for this species complex involve transloca- tions between chromosome pairs (e.g. karyomorphs 2n = 39/40D and 2n = 40/41G) (Bertollo et al., 2000), or translocation and heterochromatinization, as proposed for the evolution of the 2n = 42B karyomorph from a 2n = 42A karyomorph ancestor © 2009 The Authors Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
  • 10. PHYLOGEOGRAPHY OF HOPLIAS MALABARICUS 2335 40F Pará River 40F Pandeiros River Ibimirim Haplogroup II A 40F Pandeiros River 40F Itapicuru River 40F Curvelo River 100, 1·00, 100, 100 Ibimirim 40F Pandeiros River 40F Pará River 40F Curvelo 40F Pandeiros River 66, 1·00, 83, 71 42B Carioca Lake 42A São Mateys River 42B Dom Helvécio Lake 42 A São Mateus River 42A São Mateus River 100, 1·00, 98, 98 42B Dom Helvécio Lake 42A São Mateus River 42A Macaé River 63, 1·00, 86, 64 42A São João River 62, 0·97, *, 61 Haplogroup II B 42 A Paraíba do Sul River 42A Itabapoana River 99, 1·00, 90, 100 42A Paraiba do Sul River 42A Tombadouro Creek 42A Jacaré River Macacu 100, 1·00, 83, 100 Paranaguá 42A Ribeira River Haplogroup I 42A Tombadouro Creek Hoplias lacerdae 0·02 Fig. 6. Phylogenetic relationships of ATPase 6 haplotypes derived from Hoplias malabaricus karyomorphs. The topology was obtained with neighbour joining analysis. Bootstrap values are expressed as neighbour joining–Bayesian–maximum likelihood–maximum parsimony. The asterisk indicates a polytomy with maximum likelihood analysis. Bar = molecular distance. © 2009 The Authors Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
  • 11. 2336 U. SANTOS ET AL. (Born & Bertollo, 2000). The 2n = 40/41G karyomorph also has a large first pair of metacentrics and is therefore morphologically closest to the 2n = 40F karyomorph. Females with both karyomorphs have identical karyotypes and may be sister kary- omorphs or species. Ongoing studies including these karyomorphs, using additional chromosomal markers will allow for a more thorough hypothesis of the evolutionary origins of this sex chromosome system. C-positive heterochromatic bands were always located in the centromeric/ pericentromeric region of all chromosomes and in the telomeric region of some pairs, in addition to multiple Ag-NORs sites. These heterochromatic and NOR char- acteristics were similar to those reported for other populations or karyomorphs of H. malabaricus (Dergam & Bertollo, 1990; Haaf et al., 1993; Bertollo, 1996, 1997; Born & Bertollo, 2000; Vicari et al., 2003, 2005). In the Pandeiros River sample, Ag-NORs were restricted to telomeres of some chromosomes and appeared in larger numbers than 18S rDNA sites, but the latter also hybridized to the pericentromeric region of one chromosome pair [Fig. 3(a)]. There is currently no explanation for the apparently non-specific nature of some telomeric Ag-NORs in the Pandeiros sample, which is as large as seven in the Trˆ s Marias region (Dergam & Bertollo, 1990). On e the other hand, pericentromeric 18S rDNA sites were not visualized by the silver nitrate method (these data and Dergam & Bertollo, 1990), which may be due to preferential telomeric NOR activation, as already pointed out for other Hoplias spp. karyomorphs (Vicari et al., 2005). Hybridization sites of the 5S rDNA probe were interstitially located in a small metacentric pair in both populations, a pattern also reported for H. malabaricus samples from Trˆ s Marias (Ferreira et al., 2007). The e patterns obtained with repetitive DNA class 5SHind III-DNA were similar to those obtained for the Trˆ s Marias population (Ferreira et al., 2007). e Karyomorph 2n = 42A The karyotypes of specimens from Tombadouro Creek and Jacar´ River were char- e acterized as karyomorph 2n = 42A, a widespread karyotypic form in the Neotropics (Bertollo et al., 2000). C-banding slowed conspicuous centromeric blocks in almost all chromosomes, an overall pattern also reported by Born & Bertollo (2001) for other Grande River 2n = 42A populations. High levels of Ag-NOR variation observed in the Tombadouro Creek population have also been reported elsewhere in the Grande River basin (Born & Bertollo, 2001). Jacar´ River H. malabaricus showed Ag-NORs e restricted to one chromosome pair, characterizing the lowest number of activated NORs reported so far as a fixed character state for any H. malabaricus population. FISH with an 18S rDNA probe, however, showed a larger number of NORs cistrons in both 2n = 42A karyomorph populations. On the other hand, FISH mapping with a 5S rDNA probe revealed differences between the Tombadouro Creek and the Jacar´ River populations, where they e hybridized on a large submetacentric and a small metacentric pair, respectively. This difference contrasted with previous reports of other H. malabaricus popula- tions from the upper Paran´ Basin, the Aragu´ River, where both pairs showed a a fluorescence with this probe (Martins et al., 2006). Considering that at least some specimens from both basins shared identical haplotypes (see below), these population differences highlighted the great potential of this probe for population studies. Patterns of variation of 18S rDNA were substantially different from the ones reported by Cioffi et al. (2009). These authors indicated the presence of five sites in © 2009 The Authors Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
  • 12. PHYLOGEOGRAPHY OF HOPLIAS MALABARICUS 2337 the 2n = 42B karyomorph and four fluorescent sites in a 2n = 42A karyomorph from the upper Paran´ River basin, whereas the results of this study showed that the same a karyomorph in the Jacar´ and Tombadouro populations had three fluorescent sites. e This reduction in numbers was also evident in the 2n = 40F karyomorph, when compared with the 2n = 40C and 2n = 39/40D karyomorphs, which showed five fluorescent sites. Thus, this probe appears to be especially informative for studying among-karyomorph variation. According to the haplotype-based trees, 5SHind III-DNA patterns reflected a small increase in site numbers (from 18 to 20); Cioffi et al. (2009) also found variation in site numbers for other 2n = 42 and 2n = 40 populations. Also, the fluorescent centromeric region in the first or second chromosome pairs appears to be particularly informative. The presence of the fluorescent region in the first chromosome pair was observed in the 2n = 42A karyomorph, as is the case for 2n = 42B, 2n = 40C and 2n = 39/40D karyomorphs (Cioffi et al., 2009), representing a plesiomorphic state. On the other hand, the 2n = 40F karyomorph showed an apparently apomorphic condition, where the fluorescent region occurred in the second chromosome pair. Absence of the fluorescent site in the first chromosome pair might be attributed to a more recent origin of the first chromosome pair and its possible origin from chromosomes lacking this site. This hypothesis, however, involves a particularly complex evolutionary process, because the persistence of diploid number would also involve alterations of other chromosome pairs. Alternatively, the 2n = 40F kary- omorph might have evolved directly from a 2n = 42A ancestor, causing a reduction in diploid number and producing an independent 2n = 40 lineage. This hypothesis is consistent with the hypothesis of Bertollo et al. (2000), based on gross chromosome morphology, that the 2n = 40F, 2n = 40/41G and 2n = 42E karyomorphs represent a monophyletic group within H. malabaricus. In the submetacentric chromosome group, the pattern of fluorescent centromeric sites in three major chromosome pairs also sets apart the 2n = 40F karyomorph from other karyomorphs. The 2n = 42A karyomorph and the three karyomorphs reported by Cioffi et al. (2009) have conspicuous fluorescent centromeric regions in these pairs, while this number was reduced to two pairs in the 2n = 40F karyomorph. Homeologies among other chromosome pairs seem less reliable and must await further data. These FISH results and the presence of a unique sex chromosome system clearly indicate that the 2n = 40F karyomorph is a taxon with the largest suit of derived karyotypic characters in this species complex. M O L E C U L A R D ATA Molecular data are congruent with the cytogenetic results and suggest a long reproductive isolation between the karyomorphs. Haplogroup I included some of the 2n = 42A karyomorph haplotypes from the Tombadouro Creek and haplotypes derived from three coastal basins located south of the Para´ba do Sul River. This ı haplogroup showed low levels of within variation, suggesting that these haplotypes were closely related. Some haplogroup II haplotypes were sympatric with haplogroup I haplotypes at the Tombadouro Creek. Within haplogroup IIB, all haplotypes from the Grande River were closely related to haplotypes from four coastal basins. These haplotypes showed a sister group relationship with haplotypes from the S˜ o Mateus a and Doce coastal basins that were not represented in samples from the S˜ o Francisco a © 2009 The Authors Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
  • 13. 2338 U. SANTOS ET AL. and Grande Rivers. Some 2n = 42 trahiras from the Tombadouro Creek and Jacar´ e River shared identical haplotypes, suggesting a recent range expansion from the Grande to the S˜ o Francisco basins. a These results provide insights into the evolution of H. malabaricus karyomorphs and the history of contact between coastal and continental hydrological basins. The most inclusive study of ND2 and ATPase 6 divergence between freshwater fishes is Bermingham et al. (1997), who estimated a rate of divergence for this gene segment of 1·3% per million years. Overall sequence (p-distance) divergence of the divergent haplogroup I haplotypes versus haplogroup II haplotypes would suggest a lineage split at 4·2 million years ago, an age that is younger than the inferred Amazon–Paranean vicariance, which has been dated at 11·8–10 million bp (reviewed in Lundberg et al., 1998). This isolation left 2n = 42 and 2n = 40 kary- omorphs in both basins (Bertollo et al., 2000), indicating that karyotypic evolution was well underway during the Late Miocene Epoch. Miocene fossils are also consid- ered to be similar to present-day taxa (Lundberg, 1998). The information content of molecular variation of this species complex may be more elusive: a preliminary analysis of ATPase 6 sequences in H. malabaricus populations from the Ama- zon–Paranean boundary basins reveals two clades that show large differences in p-distance divergence within each clade (0·10 and 0·34% per million years, respec- tively) (J. A. Dergam, unpubl. data), suggesting that molecular evolution within the H. malabaricus complex may be highly variable and influenced by other causes. Fac- tors such as efficiency in DNA repair mechanism (Britten, 1986), generation time (Wu & Li, 1985), metabolic rate (Martin & Palumbi, 1993) and demographic pro- cesses (Ohta, 2002) may affect substitution rates among taxa. Hence, it is plausible to hypothesize a strong effect of demographic processes on the rates of molecular substitution in these populations, considering the close phylogenetic relationships among H. malabaricus karyomorphs and the adaptation of H. malabaricus to life in small populations, which is also reflected in their high levels of karyotypical vari- ation. Slightly disadvantageous substitutions can drift to high frequencies in small populations (Ohta, 2002), thereby altering substitution rates in the DNA. Therefore, H. malabaricus and other species with similar ecology may be particularly poor can- didates for using the molecular clock hypothesis to estimate divergence times. In the highly migratory species, Prochilodus spp., substitution rates of ATPase 6,8 range from 0·8 to 2·5% (Sivasundar et al., 2001), suggesting haplotypes in H. malabaricus are among the most divergent in the Neotropical region. The present study is the first to indicate such a deep molecular divergence within the same karyomorph, although deep cytochrome b divergences were also observed among populations of the catfish Pimelodus albicans (Valenciennes) in the River Plate basin (Vergara et al., 2008). The location where lineage splitting occurred is limited to the distribution of current karyomorphs. The Paran´ River basin harbours at least three karyomorphs, a whereas the 2n = 40F karyomorph appears to be widespread in the S˜ o Francisco a River basin and the 2n = 42A karyomorph is apparently restricted to the Par´ River a headwaters. North-eastern coastal populations harbour the 2n = 40F karyomorph as far as the Buranh´ m River to the south (J. A. Dergam, unpubl. data) (Fig. 1), e and these haplotypes had the lowest degree of divergence. This macro-geographical pattern suggests that lineage splitting occurred elsewhere and not in the relatively isolated S˜ o Francisco River basin. In haplogroup IIB, the close relationship between a the 2n = 42A and 2n = 42B karyomorphs indicated that karyotypical differentiation © 2009 The Authors Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
  • 14. PHYLOGEOGRAPHY OF HOPLIAS MALABARICUS 2339 involved few alterations that resulted in a differentiated XX/XY chromosome system (Bertollo et al., 2000). S T R E A M P I R AC Y On a local scale, the apparently restricted range of the Tombadouro Creek 2n = 42A karyomorph may have resulted from stream piracy from a former Jacar´ River e tributary. The Tombadouro Creek is 12 km from the Jacar´ across the Galga water- e shed. Three hypotheses might explain this dispersal. First, boundary crossing may have resulted from either human mediated activities. Second, dispersal may have occurred through a high-altitude wetland that used to drain into both basins. For example, the connection may have been destroyed by the construction of BR 494 Highway, which is 8 km away at its closest point from the Tombadouro Creek. Third, headwater capture in the region may have occurred by relatively recent reactivation of ancient faults (Saadi et al., 2002). Altitudinal characteristics are also consistent with the direction of stream capture. Tombadouro is at 860 m altitude, whereas the Jacar´ River is at 1038 m. This area is within the range of a continental fault known e as the Upper Grande River Crustal Discontinuity (Saadi et al., 2002). This fault represents the southern limit of the S˜ o Francisco craton and the movable belts of a southern Minas Gerais State (Fig. 1) and has been active in the last 15 000 years. The phylogenetic origins of two highly divergent haplotypes in the Tombadouro Creek became clear only after haplotypes from coastal basins were included in the analysis. Most critically, haplotypes found in sympatry in the S˜ o Francisco River a are related to haplotypes in different coastal basins. Under the allopatric model of speciation, this asymmetry suggests that ancestral fish dispersed from the coastal to the continental drainages, and the present study is the first to indicate unambiguously the direction of this dispersal. The timing of the dispersal between coastal and inland drainages, however, could not be estimated, because molecular clock expectations are not suitable for H. malabaricus. Therefore, the phylogeographic history of this group does not fit any of the three phylogenetic patterns proposed by Ribeiro (2006). On the other hand, low genetic divergence between the S˜ o Francisco and Itapicuru River a drainages may be part of a recent faunal exchange in the arid region of north-eastern Brazil that is apparent in many other species of fish (Rosa et al., 2004). Ribeiro (2006) indicated that the Atlantic drainages (Doce, Para´ba do Sul and ı Ribeira) have existed since the break-up of Gondwana. The results of the present study suggested that populations in these basins represent three biogeographic units, for which only two have close representatives in the boundaries of the Jacar´ and Par´ e a drainages. Dergam et al. (2002) previously reported a close phylogenetic relatedness between two haplotypes in the Doce and lower Grande River basins. Fish dispersal from coastal to continental drainages may have been restricted to only a few taxa adapted to headwater conditions, because coastal drainages are characterized by high levels of endemism (Ribeiro, 2006) and the Proterozoic Mantiqueira Range is con- sidered to be an efficient barrier between coastal and continental drainages (Ingenito & Buckup, 2007). To the south, stream piracy has been particularly intensive in the Para´ba do Sul, Ribeira and Upper Paran´ basins (Ribeiro, 2006). ı a The present study provided important information on the population distribution of H. malabaricus in S˜ o Francisco River basin and on historical relationships with a populations in the Grande River and coastal basins. The 2n = 40F karyomorph and © 2009 The Authors Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
  • 15. 2340 U. SANTOS ET AL. its associated haplotypes were widely distributed throughout the S˜ o Francisco basin, a maintaining an apparently stable karyotypic structure of the chromosomal markers analysed here. This karyomorph was characterized by a XX/XY sex chromosome system. In contrast, the 2n = 42A karyomorph showed a more restricted distribution in S˜ o Francisco basin and was closely associated with populations in a Grande a River basin tributary, where the 2n = 42A karyomorph predominated. The 2n = 42A karyomorph populations isolated in drainages, however, showed striking differences for some markers, notably the Ag-NORs and 5S rDNA sites. The patterns of variation in repetitive DNA sequences as revealed by 5S rDNA probes indicated that these markers appeared to be useful population markers, showing significant differences among localities and karyomorphs. Patterns of 5SHind III-DNA variation yielded the largest phylogenetic signal and placed the 2n = 40F karyomorph in a high position in the phylogeny of karyomorphs that occur in eastern Brazil. Given the current status of phylogenetic data, an understanding of distribution patterns for H. malabaricus will be particularly challenging. Studies of the distri- bution patterns of freshwater fishes have considered endemism (Vari, 1988), faunal composition including endemic and widespread species (Hubert & Renno, 2006), molecular data (Sivasundar et al., 2001; Montoya-Burgos, 2003) or a combination of morphological and molecular data (Menezes et al., 2008). When integrated into a wider geographic context, molecular and cytogenetic patterns can be informative of faunal dispersals between drainages. The results of the present study suggested that recent fish dispersal may occur even within apparently stable geological regions and that headwater stream piracy between coastal and continental (upper Paran´ tribu- a taries) basins may have played a role in producing the high levels of freshwater fish diversity in the Neotropics. Finally, the topology of mtDNA-based tree suggests that the proposal of Dergam & Bertollo (1990) to separate species by diploid numbers would result in a paraphyletic arrangement, in which some Hoplias spp. haplotypes associated with 2n = 42 kary- omorphs would be more closely related to trahiras with 2n = 40 karyomorphs than to other fish with 2n = 42 karyomorphs. Although haplogroup I appeared as the sis- ter group of the remaining trahiras, haplogroup II is less supported and the possible ancestry of karyomorphs must wait for more data. A time frame for dispersal between coastal and continental drainages is elusive for the H. malabaricus species complex. Nevertheless, geographical isolation during coastal diversification has enhanced spe- ciation processes and genetic isolation between karyomorphs, a process that has resulted in molecular and cytogenetic differentiation between populations. These results are consistent with the existence of multiple independent lineages that can be easily detected with standard cytogenetic techniques. Although Hoplias spp. are common members of lowland freshwater communities (Bonetto et al., 1969; Winemiller, 1991), at least part of their widespread distribution in the Neotropics is due to their ability to colonise high-altitude headwaters, such as in the Jacar´ and Tombadouro localities. The question of how this non-migratory e sedentary species has adapted to sluggish waters and to waters at high altitudes is not explained by current geological data. Nevertheless, this broad ecological diversity indicates a long history of Hoplias spp. in south-eastern Brazil. J. T. da Costa, J. B. Santos, R. de F. Guimar˜ es and A. M. R. de Souza provided support a during fieldwork. W. Evangelista and U. Jacobina collected samples from the Ibimirim lakes © 2009 The Authors Journal compilation © 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 75, 2326–2343
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